1. Field of the Invention
This invention relates generally to mechanisms of gene expression in plants and more specifically to regulation of expression of genes in plants in a "floral-preferred" manner. Regulation of expression is achieved using at least one of several transcriptional regulatory units capable of driving expression of genes within floral tissues of a plant. Said transcriptional regulatory unit will ultimately be utilized for driving expression of genes that confer a selective advantage to plants.
2. Description of the Related Art
Over the past decade, the valuable method of introducing foreign genes into plants has been used to study promoter strength and tissue-preferred gene expression (Benfey and Chua, 1989). Despite prolonged and substantial effort by many laboratories, development of genetic transformation techniques for maize has been difficult to achieve (Gordon-Kamm et al., 1990). To understand the mechanisms regulating tissue-preferred expression and the cis-acting factors interacting with tissue-preferred elements, a necessary step is to define the promoter regions controlling expression. Transgenic plants are a useful tool in such studies. In general, these types of studies have not been possible using transgenic maize plants because of the absence of a routine transformation system (Kyozuka et al., 1994). This invention illustrates the feasibility and importance of using transgenic maize in the study of promoter regulation in a homologous system. Transcriptional control elements which drive "tissue-general" or "constitutive" gene expression in plants have been described. These include the promoters of the Agrobacterium nopaline synthase gene (Depicker, et al. 1982) and the maize ubiquitin gene (Christensen, et al. 1992). Other promoters have been well characterized and utilized for driving constitutive gene expression in transgenic plants e.g., CaMV 35S (Odell et al. 1985)!. There exists both an increasing interest in co-transforming plants with multiple plant transcription units and a realization of several potential problems associated with this technique. In order to protect plants from certain pests, pathogens, adverse weather conditions or to provide growth or other survival advantages to a plant, it is useful to direct gene expression to certain tissues of a plant. In this manner, gene expression may be maintained at a low or non-existent level in tissues in which expression of said gene could prove detrimental to the plant or may result in a drain on the plant's energy resources. It is, therefore, considered important by those skilled in the art to develop transcriptional regulatory units (including but not limited to promoters, enhancers and repressors) useful in limiting gene expression to certain tissues of a plant.
The P gene encodes a myb-like transcription activator, controlling phlobaphene pigmentation in maize floral organs by directly activating a flavonoid biosynthetic gene subset (Grotewold et al., 1991 & 1994). The floral tissues in which the P gene is expressed include but are not limited to kernel pericarp, the lemma, palea and glumes of the female flower, and similar organs of the male flower. Due to its conspicuous red pigmentation phenotype, the P gene has been the object of extensive genetic analysis since the pioneering work of Emerson (1917). The maize P alleles are usually named based on pigmentation in these two tissues, e.g., P-rr: red pericarp and red cob; P-wr: white pericarp and red cob; P-rw: red pericarp and white cob; P-ww: white pericarp and white cob. Despite the extensive and long-standing genetic studies of the P gene, little is known regarding the mechanism of P gene regulation of tissue-preferred phlobaphene pigmentation in certain floral tissues (Styles, & Ceska, 1977). The P-vv allele, which specifies variegated pericarp and cob pigmentation and contains the transposable element Ac inserted in the P-rr allele (Lechelt et al., 1989), has been used to study Ac transpositional mutagenesis (Athma et al., 1992) and the transpositional mechanisms (Chen et al., 1987 and 1992). Molecular mapping and DNA sequence analyses have shown that reinsertions were clustered in two regions, the 1.3 kb sequences immediately 5' of the transcription start site and an upstream region corresponding to a 1.2-kb SalI fragment, localized 4853 bp upstream of the TSS (Moreno et al., 1992). Although the insertions in the 1.2-kb SalI fragment are approximately 5 kb upstream from the TSS, a lightly to very lightly variegated phenotype is observed in plants with such insertions. It was suggested that these insertions might affect the activity of cis-acting sequences, such as enhancer elements required for P-rr activity. If such distal enhancers exist, the P-rr promoter would represent the largest plant promoter reported to date (Moreno et al, 1992). A new allele, P-pr, was found to arise from epimutation of P-rr (Das and Messing, 1994). P-rr specifies a red pericarp and red cob glumes and P-pr specifies patterned pericarp and red cob. Reduction in red pigmentation of plants expressing P-pr was associated with decrease in P-pr mRNA levels, possibly due to greater methylation in the promoter or elsewhere in the P-pr gene. The previously mentioned upstream 1.2 kb region has been demonstrated to affect expression of the P-rr gene. Alteration of the 1.2 kb region have been shown to include insertions, methylation, and tissue-specific changes in chromatin structure. It was therefore hypothesized that this region may contain cis-acting elements important to the tissue-specific pattern of expression observed in plant tissues (Lund et al., 1995).
To understand the regulatory role of the upstream 1.2-kb SalI region and to determine which regions of the P-rr promoter direct floral specificity to the P-rr gene, we tested DNA constructs comprising regions of the P-rr promoter operably linked to a reporter gene, the b-glucuronidase gene (GUS), in transient assays (Martin, T., et al. In S. R. Gallagher (ed.), GUS Protocols: Using the GUS gene as a reporter of gene expression, p. 23-43). These constructs were also tested by transformation of plant cell cultures and the subsequent generation of stable transgenic plants. It is demonstrated that the primary determinants of maize P-rr floral-specificity resides in the basal 500 bp region immediately 5' of the transcription start site (TSS). Tissue specificity and a precise developmental pattern of P gene promoter-driven GUS gene expression in stable transgenic maize was observed in floral tissues including pericarps, cob glumes, silk, and husks without any detectable expression in roots, stems, and leaves. Gene expression driven by this region of the promoter, while floral-preferred, is at low levels. Expression vectors comprising preferred regions of the P promoter were constructed and certain regions demonstrated to function as enhancer elements. The enhancer elements are separated by up to 3.6 kb and possibly function as long distance enhancer elements. The results of the functional assays are consistent with predictions from Ac insertional mutagenesis experiments (Moreno et al., 1992), P-pr methylation pattern (Das and Messing, 1994), and DNase I sensitivity assays (Lund et al., 1995). These data underscore the importance of these sequences for P-rr expression.
There is a need in the art for novel transcriptional regulatory elements which are capable of driving floral-preferred gene expression in plants. It is considered important by those skilled in the art to continue to provide tissue-preferred transcription units capable driving expression of genes that confer resistance to plant pathogens, pests, herbicides, or adverse weather conditions including but not limited to cold, heat, and flooding as well as genes which influence growth of or yield from said plants. The inventions described within this application may be utilized to drive floral-preferred gene expression in plants, and therefore, are considered important to those skilled in the art.